Cloning of the human insulin receptor cDNA has revealed that there are two different naturally occurring forms of the insulin receptor. These are generated by alternate splicing of a 36 nucleotide exon to yield receptors with or without a 12 amino acid insert near the COOH-terminus of the alpha-subunit. The two alternate forms of the insulin receptor are expressed in a tissue-specific and developmentally regulated manner, suggesting that the difference may be functionally significant. This proposal is to express the cDNAs encoding these alternate forms in host cells to generate stable cell lines with one or the other receptor in comparable numbers. Preliminary data indicate that the receptors differ in their affinities for insulin and endocytotic itineraries. This will be confirmed in independent clones of transfected cells. The properties of the receptors in vitro and in vivo will be analyzed. Studies will include ligand binding and competition of ligand by insulin, insulin-like growth factor I, and proinsulin in order to establish the affinities and specificities of the two receptors. Tyrosine kinase activity will be assessed by measuring autophosphorylation and substrate phosphorylation as functions of time and insulin concentration, using partially purified receptors as well as in intact cells. The intracellular itinerary of the receptor subtypes will be investigated in the transfected cells by measuring initial endocytotic rates and degradation rates of insulin as well as endocytosis, recycling, and down regulation of the receptors themselves. The ability of the receptors to mediate insulin-stimulated hexose uptake, activation of glycogen synthase and mitogenesis will be assessed by dose-response analysis in the transfected cells. The physiologic consequences of co-expression of both receptor forms on insulin action will also be studied. The hypothesis that changes in insulin receptor affinity seen in some cells after down-regulation or corticosteroid treatment may be due in part to altered regulation of the splicing event will be tested using the polymerase chain reaction technique. Finally, various cells and tissues from subjects with non-insulin dependent diabetes mellitus and normal controls will be tested for possible alterations in the relative expression of the receptor subtypes.